Although bacillus Calmette-Guerin (BCG) is the most effective adjuvant treatment for human superficial transitional cell carcinoma (TCC) of the bladder, it is ineffective in up to one third of the cases and it is associated with serious toxicity. The goals of this proposal are to improve the efficacy and reduce the toxicity of BCG by genetically engineering BCG to express and secrete powerful immunoregulatory cytokine proteins. This proposal has three specific aims: Specific Aim #1: To engineer recombinant BCG (rBCG) to express and secrete immunoregulatory proteins. Novel genes will be introduced into BCG using a plasmid-based transfection system developed. Specific cytokine genes have been selected based on their potential to complement ongoing cytokine responses (TNF-alpha), antagonize specific cytokine responses (IL-4, IL- 10), or recruit accessory antitumor mechanisms (GM-CSF, IFN-alpha, IL-4). In addition, the Lac Z (beta-galactosidase) gene will be used as a reporter gene to track and quantify rBCG during in vitro and in vivo use. The Herpes Simplex Virus I-thymidine kinase (HSV-tk) gene will be evaluated as a potential "suicide" gene to eradicate rBCG using the anti- herpetic drug acyclovir. Specific Aim #2: To improve our understanding of cytokine pathways elicited by rBCG that have the capacity to modulate antitumor responses. It is important to investigate the cytokine pathways elicited by rBCG so that rational strategies can be formulated to maximize cellular and cytokine responses beneficial to the elimination of TCC while minimizing unproductive or deleterious responses. Endogenous cytokine production from host splenocytes stimulated by rBCG will be measured using sensitive ELISA assays and semi-quantitative Northern blotting. Particular cytokine-BCG combinations will be identified that antagonize or enhance specific splenocyte responses. The specialized immune cell types responsible for these responses will be determined by depleting splenocytes of specific subsets or by replacing them with splenocytes derived from animals with well defined functional immune defects. Specific Aim #3: To assess the ability of cytokine rBCG to enhance the immunotherapy of experimental bladder cancer in animal models. Cytokine rBCGs will be tested for activity against TCC in both mouse and rat tumor models. The rat TCC model will provide an independent assessment of rBCG efficacy against the most clinically significant form of bladder cancer, carcinoma-in-situ (CIS).